This application is a 371 of PCT/GB99/01902.
The present invention relates to plant modification, especially modification by manipulating the activity of a combination of plant enzymes having starch synthase activity to alter the nature of starch obtainable from the plant. In particular, it relates to manipulation of activity of the plant enzymes starch synthase II (SSII) and starch synthase III (SSIII), modified plants obtained thereby and their use as food products. The invention also relates to starch having novel properties and to uses thereof.
Starch is of importance in a variety of food and other applications, such as in the paper, textiles and adhesives industries. Commonly, native starches obtained from storage organs of plants such as cereal endosperms, potato tubers and pea embryos are further modified, generally by either chemical or physical means, to produce starches having improved properties more suited to the intended application. Commercial interest has been directed, in particular, to methods for modifying or manipulating the temperature at which onset of gelatinisation occurs (that is, the process of collapse or disruption of molecular order within the starch granules when aqueous suspensions of starch granules are heated, causing the granules to swell and absorb water). Methods for manipulating the gelatinisation temperature of starch which have been described in the literature include the use of additives (see, for example, Evans et al, Starke, 34, 224-231, 1982) or chemical or physical pre-treatments (see Stute, Starke, 44, 205-21, 1992).
It would be desirable commercially to provide plants which intrinsically produce starches having the desired properties, thereby obviating the need for additional, costly and generally inefficient, modification steps. To this end, considerable interest has been expressed in the art in studying the starch biosynthetic pathway in plants, more particularly in the potato, with the aim of modifying the plant genome to produce starches with novel and advantageous properties.
Approaches to modifying the starch biosynthetic pathway in plants using recombinant DNA technology have recently been described in the literature. In particular, methods based on manipulating the activity of plant enzymes having either starch branching or starch synthase activity, generally regarded as the most important starch-synthesising enzymes, have been studied.
WO 96/34968, published November 1996, discloses a nucleotide sequence encoding an effective portion of a Class A starch branching enzyme (SBE) obtainable from potato plants, which sequence can be introduced, conveniently linked in an antisense orientation to a suitable promoter and preferably together with an effective portion of a sequence encoding a Class B starch branching enzyme, into a plant to alter the characteristics of the plant. It is disclosed that starch extracted from a plant so transformed has a viscosity onset temperature (which gives an indication of the onset of the gelatinisation process) which is elevated by around 10 to 25xc2x0 C. compared to starch extracted from a similar but unaltered plant.
Isoforms of starch synthase are found in the storage organs of most species of plants and there is currently much interest in characterising them and studying their role in starch synthesis. In pea for instance, the low amylose locus (lam) was shown to be a mutation in the granule-bound starch synthase I (GBSS I) gene (Denyer et al Plant Cell Environ. 18,1019-1026, 1995) and very recently, the rug5 locus was shown to be a mutation in the major soluble isoform of starch synthase (SSII) (Craig et al Plant Cell 10, 413-426, 1998). In maize, the waxy gene encoding the granule-bound starch synthase I (GBSS I) has been cloned (Shure et al Cell 35, 225-233, 1983) and the recent cloning of the dullI gene identifies that locus as a starch synthase, most likely SSII (Gao et al Plant Cell 10, 399-412, 1998). In addition, several recent patent publications describe the cloning of starch synthases from maize (WO 97/44472, WO 97/20936) and wheat (WO 97/45545). The role of each isoform in the control of starch synthesis and structure is unclear at present since the contribution of each isoform to the total activity varies considerably between species.
Marked effects on the properties of starch, in particular a reduction in the viscosity onset temperature compared to untransformed material, have been observed when potato plants are modified by manipulation of one particular starch synthase enzyme, now designated as starch synthase III (SSIII) (see EP-A-0779363, National Starch and Chemical Investment Holding Corporation, published June 1997, and Marshall et al, Plant Cell, 8, 1121-1135, 1996). Here, a reduction in the onset temperature for gelatinisation of starch extracted from transformed potato plants of at least 5xc2x0 C. compared to starch extracted from equivalent, non-transformed plants was reported. In addition to the differences in starch properties, altered starch granule morphology and reductions in soluble starch synthase activity in the order of 80% were reported.
WO 96/15248 (Institut Fur Genbiologische Forschung Berlin GmbH), published May 1996, discloses potato plants transformed with a portion of either one of two cDNA clones, denoted SSSA and SSSB, which are said to encode isoforms of potato soluble starch synthases.
Starch synthase III (SSIII), a largely soluble isoform having a molecular mass as judged by SDS-PAGE in the range of 100-140 kDa is one of three isoforms of starch synthase, each encoded by a different gene, which have been purified in developing potato tubers. The other isoforms which have been described and characterised are a granule bound starch synthase of approximately 60 kDa, designated granule bound starch synthase I (GBSSI, also known as the waxy protein), see, for example, Hovenkamp-Hermelink et al, (Theor. Appl. Genet., 7, 217-221, 1987), and starch synthase II (SSII, formerly known as GBSSII), see, for example, Edwards et al, (Plant J., 8, 283-294, 1995) and Marshall et al (above), which isoform is found in both soluble and granule bound forms and has an apparent molecular weight of approximately 78 kDa. Potato plants either lacking these other isoforms or having reduced isoform activity have been generated and the effects on the properties of the starch obtained therefrom have been studied.
Elimination of GBSSI activity through mutagenesis resulted in a tuber starch with no amylose whereas reduction of its activity through expression of antisense RNA leads to tuber starches with reduced levels of amylose. The physical properties of the starches so produced are similar to those low- or zero-amylose starches known from other sources such as the waxy mutants of cereals (see, for example, Flipse et al, Theor. Appl. Genet., 92, 121-127, 1996).
Reductions in the amount of both soluble and granule-bound SSII protein via expression of antisense RNA were found to have little or no effect on the total (soluble and granule-bound) starch synthase activity of the tuber, the amount of starch, or the amylose to arnylopectin ratio of the starch (see Edwards et al, 1995 and Koxcex2mann et al Macromol. Symp. 120, 29-38, 1997). The present inventors have also found that there is little effect on the physical properties of the starch from such tubers.
There remains a continuing need for the development of improved methods for modulating or manipulating starch biosynthesis in plants with the aim of producing transformed plants and starches having improved properties. In particular, there is considerable commercial interest in the development of improved methods for producing transformed plants providing starches having a reduced viscosity onset temperature compared to currently available starches as this would be advantageous in allowing for the use of milder processing conditions and reduced energy use. Benefits resulting from the use of such plants in the preparation of food products include improvements in food quality, reduction of off flavours or volatiles and improvements in colour.
Accordingly, in a first aspect the invention provides a method of modifying one or more characteristics of a plant comprising introducing into the plant a combination of sequences, each sequence comprising a gene encoding an enzyme having starch synthase activity, or a sequence functionally equivalent thereto or an effective part thereof, each sequence being operably linked to a promoter so as to inhibit the expression of corresponding endogenous genes in the plant. The introduced sequences may inhibit the expression of the corresponding endogenous genes in the plant, either by means of sense suppression, or by means of antisense inhibition, both of which phenomena are known to those skilled in the art. The introduced sequences may inhibit the expression of the corresponding endogenous genes in the plant at the nucleic acid (DNA and/or RNA) and/or amino acid level. Typically the combination comprises sequences encoding two starch synthase genes, or sequences functionally equivalent thereto.
Generally, the characteristic of the plant altered by the method will include the level of activity of one or more starch synthases in the plant. In addition the physical and/or chemical properties of starch produced by the plant may be altered.
In another aspect, the invention provides a plant which has been modified by the method defined above and its use in the preparation of a food product.
Progeny of a plant according to the invention and any part thereof are also provided.
The invention also provides starch obtained or obtainable from a plant according to the invention or the progeny of such a plant and the use thereof, for example in the preparation or processing of foodstuffs, or in the paper, textile or adhesive industries. In a further aspect, the invention provides a method of modifying starch comprising modifying a plant by the method defined above and extracting therefrom starch having modified properties compared to starch extracted from equivalent, but unmodified, plants.
The invention further provides a DNA construct comprising a combination of sequences, each sequence comprising a gene encoding an enzyme having starch synthase activity, or a functionally equivalent sequence thereof or an effective part thereof, each sequence being operably linked to a promoter. Plants comprising such a construct or any part of the progeny thereof are also included within the scope of the invention.
As used herein, xe2x80x9cplantxe2x80x9d means a whole plant or part thereof, or a plant cell or group of plant cells. Parts of a plant typically include storage organs, such as potato tubers, cereal endosperms or pea embryos and seeds.
A xe2x80x9cgenexe2x80x9d is a DNA sequence encoding a protein, including modified or synthetic DNA sequences or naturally occurring sequences encoding a protein, and excluding the 5xe2x80x2 sequence which drives the initiation of transcription.
A sequence functionally equivalent to a sequence encoding an enzyme having starch synthase activity includes any sequence which, when placed under the control of a suitable promoter, inhibits the expression of the corresponding, naturally occurring endogenous gene. Functionally equivalent sequences are intended to include those sequences exhibiting at least 60% nucleotide homology, preferably at least 80%, more preferably at least 90% homology with the sequence of a naturally occurring endogenous plant starch synthase gene. Such equivalent sequences are generally able to hybridise under standard laboratory conditions (see for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, hereinafter xe2x80x9cSambrookxe2x80x9d) with the xe2x88x92ve strand of a naturally occurring endogenous plant starch synthase gene.
Preferred starch synthase genes for use in the method of the invention are the starch synthase II gene from potato (Edwards et al., 1995) and the starch synthase III gene from potato (Abel et al., Plant J. 10, 9810991; Marshall et al., 1996). Functional equivalents of the potato SSII gene have been described for pea (Dry et al., 1992 Plant J. 2, 193-202); cassava (Munyikwa et al., 1997 Euphytica 96, 65-75) and maize (Harn et al., 1998 Plant Mol. Biol. 37, 639-649). Functional equivalents of the potato SSIII gene have been described for maize (Gao et al., 1998 Plant Cell 10, 399-412) and pea (Craig et al., 1998 Plant Cell 10, 413-426; Tomlinson et al., 1998 Planta 204, 86-92).
In addition, functionally equivalent sequences particularly include those which are antisense equivalents of the sequence encoding an enzyme having starch synthase activity. Such antisense equivalents are therefore generally able to hybridise with the sense (+ve) strand of a naturally occurring endogenous plant starch synthase gene.
xe2x80x9cOperably linked to a promoterxe2x80x9d means that the sequence is positioned or connected to the promoter in such a way to ensure transcription of the sequence. The promoter is any sequence sufficient to allow the DNA to be transcribed. After the gene and promoter sequences are joined, upon activation of the promoter, the gene will be expressed.
An xe2x80x9cequivalent, unmodifiedxe2x80x9d plant is a plant which has a substantially identical genotype to a modified plant of the invention excepting the introduced sequences present in the plant of the invention. Also included within this definition are plants comprising the introduced sequences but which do not exhibit reduced starch synthase activity.
A xe2x80x9cconstructxe2x80x9d is a polynucleotide comprising nucleic acid sequences not normally associated in nature.